A genomic approach towards an understanding of clonal evolution and disease progression in Multiple Myeloma

Abstract

Multiple myeloma (MM) is a largely incurable haematological malignancy characterised by the aberrant proliferation of malignant plasma cells (PCs) in the bone marrow (BM). Next generation sequencing (NGS) studies have shown that MM patients display complex mutational landscapes involving intraclonal genetic heterogeneity. While intraclonal heterogeneity is now an established feature of MM, the genomic changes and tumour evolution associated with the transformation from the asymptomatic disease stages of Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smouldering Multiple Myeloma (SMM) to MM remains unknown. This thesis presents a unique assessment of the genomic architecture and subclonal evolution associated with the natural history of disease transformation, with the analyses of a rare cohort of paired BM samples from patients when first diagnosed with MGUS or SMM, who later went on to develop MM (n = 10). Whole exome sequencing (WES) and bioinformatic analyses identified that clonal heterogeneity was present at the asymptomatic MGUS/SMM stages of disease, with a changing spectrum of acquired mutations associated with transition to MM. Subclonality was observed at MGUS/SMM, with the presence of between 5 to 11 subclones. The progression to MM was characterised by a prevailing model of subclonal evolution defined by clonal stability, where the transformed PC subclones of MM were already present at the MGUS/SMM stage. RNA sequencing (RNAseq) revealed that the patterns of expressed genes at MGUS/SMM to MM were found to be relatively homogeneous. Moreover, RNAseq revealed that mutant genes identified by WES were generally not expressed, expressed at low levels, with most genes showing wild-type expression. Analysis of the methylome was carried out using whole genome bisulphite sequencing (WGBS). Significant hypomethylation was observed in PCs recovered at all disease stages (MGUS, SMM and MM) compared to normal PCs. Interestingly, the degree of hypomethylation observed at MGUS was maintained with progression to SMM and MM stages. In addition, the phenomenon of RNA editing in SP140, a recurrently mutated gene in human MM patients, was investigated in the 5TGM1 MM PC line. A high impact C>T (ie. U) RNA editing change was identified in exon 2 of Sp140, resulting in an early STOP codon, which was hypothesised to result in the formation of truncated Sp140 protein that may contribute to MM pathogenesis. In mouse cell lines, Sp140 RNA editing was not restricted to the 5TGM1 cell line, but editing was not observed in any human MM PC lines. CRISPR-Cas9 mediated mutation of the mouse Apobec1 and Apobec3 genes, showed that neither of these cytidine deaminases were responsible for this RNA editing phenomenon. These studies show that MGUS/SMM patients, that progress in a short time frame, appear to be sufficiently genetically complex to be on the threshold of transformation to MM. Furthermore, the intrinsic genomic complexity of MM is present at the asymptomatic stages of disease (MGUS and SMM), suggesting that extrinsic factors from the tumour microenvironment play an important role in mediating progression. Indeed, these studies suggest that early intervention at MGUS/SMM may be possible to prevent progression and result in durable cure for patients.